Screen for sodium channel modulators

ABSTRACT

A method or screen for assessing the potential of a compound to treat a pathological condition, such as arrhythmia, which is manifested by an increased late sodium current in a heart is disclosed. The method employs a mutant sodium channel protein having an amino acid sequence in which one or more amino acids among the ten amino acids occurring at the carboxy end of the S6 segments of D1, D2, D3 or D4 domains of mammalian Nav1 differs from the amino acid in wild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosine or cysteine. Cells transfected with a nucleic acid that encodes a mutant mammalian Nav1 protein, as well as isolated nucleic acid comprising a nucleotide sequence that codes for a mutant mammalian Nav1 protein are disclosed.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made under grant number 5RO0HL6607602 from theNational Heart, Lung and Blood Institute. The government may havecertain rights in the invention.

FIELD OF THE INVENTION

[0002] The invention relates to a method for screening compounds for useas anti-arrhythmic agents. The method employs a. cell line thatexpresses a mutant sodium channel protein.

BACKGROUND OF THE INVENTION

[0003] Mammalian voltage-gated sodium channels are pore-forming membraneproteins responsible for the initiation and propagation of actionpotentials in excitable membranes in nerve, skeletal muscle and heartcells. The controlled gating of sodium channels in response to membranedepolarizations is necessary for normal electrical signaling andestablishing of intercellular communication. Voltage-gated Na+ ionchannels consist of one large α-subunit (about 200 kDa) and one or twosmaller β-subunits. The α-subunits are designated “Nav” (Na for sodiumchannel and v for voltage-gated), followed by a numbering system for theparticular isoform. The Na+ channel α-subunit isoforms contain fourhomologous repeated domains (D1-D4) each with six transmembrane segments(S1-S6). The α-subunit protein alone forms a functional channel whenexpressed in mammalian expression systems. The four repeated domains arehypothesized to assemble as a peudotetrameric structure with thepermeation pathway situated at the center. FIG. 1 is a cartoon depictingone conceptualization of how the Nav protein arranges itself withrespect to the membrane. The cartoon is not accurate; it is an expandedmodel that does not attempt to depict how the four S6 segments cometogether to form the sodium channel, but it facilitates an understandingof how the proteins might align with respect to the inside and outsideof the excitable membrane. In fact, recent studies suggest that four S6C-termini may jointly close the voltage-gated cation channel at thecytoplasmic side, probably as an inverted teepee structure.

[0004] Several pieces of evidence suggest that S6 segments are involvedin Na+ channel gating. First, a number of receptors for varioustherapeutic drugs and neurotoxins such as local anesthetics (LAs),antiarrhythmics, anticonvulsants, antidepressants, pyrethroidinsecticides, batrachotoxin (BTX), and veratridine, are situated at themiddle of multiple S6 segments. Upon binding, these ligands exert theirpharmacological actions on the Na+ channel, presumably in part via theircorresponding S6 receptor. In particular, BTX drastically modifies Na+channel activation, fast inactivation, and slow inactivation, suggestingthat its receptor is linked to these gating processes.

[0005] The invention herein described arose from a hypothesis that S6segments may be structurally geared for channel activation bylateral/rotational movement via a flexible gating hinge, a glycine orserine residue located at the middle of the inner Na+ channel S6segments. This gating hinge could have two different conformations. Oneis in its relaxed straight α-helical form, which closes the channel atthe S6 C-terminal end, and the other is the bendable α-helical form,which may bend outward at a 30° angle and thus splay open the channel atthe S6 constricted C-terminus. After channel activation, S6 segments maythen form the docking site for the fast-inactivation gate. A putativeNa+ channel inactivation gate has been delineated at the intracellularlinker between D3 and D4 by West et al. [Proc. Natl. Acad. Sci. USA89:10910-10914 (1992)]. This linker could be situated at the C-terminiof S6 segments, where the inactivation gate may plug the open channelwhile it binds to its docking site. This plugging mechanism has recentlybeen demonstrated in voltage-gated K+ channels [Zhou et al., Nature411:657-661 (2001)]. The foregoing hypothesis is useful because itprovides a framework for interpreting the results and makingpredictions. However, it is important to note that the invention isbased on the results, not the hypothesis, and the hypothesis should notbe viewed as a limitation on the claimed invention.

[0006] There is very close homology among the S6 segments of mammalianNav proteins so far identified. This homology extends both throughspecies and through isoforms of the Nav protein. As can be seen in thecomparison below, the few variations that exist among the amino acids inthe amino terminal portion of the S6 segments are very conservativereplacements, and the carboxy terminal 11 amino acids of the S6 segmentsof all four domains are identical for rats and humans for both of themuscle sodium channel proteins Nav1.4 and Nav1.5: D1S6 1 6 11 16 21 26human Nav1.1 YMIFF VLVIF LGSFY LINLI LAVVA MAY (SEQ ID NO.: 1) Nav1.2YMIFF VLVIF LGSFY LINLI LAVVA MAY (SEQ ID NO.: 2) Nav1.3 YMIFF VLVIFLGSFY LINLI LAVVA MAY (SEQ ID NO.: 3) Nav1.4 YMIFF VVIIF LGSFY LINLILAVVA MAY (SEQ ID NO.: 4) Nav1.5 YMIFF MLVIF LGSFY LVNLI LAVVA MAY (SEQID NO.: 5) Nav1.8 YMIFF vVvIF LGSFY LVNLI LAVVA MAY (SEQ ID NO.: 6)Nav1.9 YMIFF VVVIF LGSFY LINLI LAVVA MAY (SEQ ID NO.: 7) rat Nav1.4YMIFF VVIIF LGSFY LINLI LAVVA MAY (SEQ ID NO.: 8) Nav1.5 YMIFF MLVIFLGSFY LVNLI LAVVA MAY (SEQ ID NO.: 9) Nav1.6 YMIFF MLVIF VGSFY PVNLILAVVA MAY (SEQ ID NO.: 10) Nav1.7 YMVFF VVVIF LGSFY LVNLI LAVVA MAY (SEQID NO.: 11) Nav1.8 YMVFF MLVIF LGSFY LVNLI LAVVA MAY (SEQ ID NO.: 12)D2S6 1 6 11 16 21 26 human Nav1.1 CLTVF MMVMV IGNLV VLNLF LALLL SSF (SEQID NO.: 13) Nav1.2 CLTVF MMVMV IGNLV VLNLF LALLL SSF (SEQ ID NO.: 14)Nav1.3 CLIVF MLVMV IGNLV VLNLF LALLL SSF (SEQ ID NO.: 15) Nav1.5 CLLVFLLVMV IGNLV VLNLF LALLL SSF (SEQ ID NO.: 16) rat Nav1.4 CLTVF LMVMVIGNLV VLNLF LALLL SSF (SEQ ID NO.: 17) Nav1.5 CLLVF LLVMV IGNLV VLNLFLALLL SSF (SEQ ID NO.: 18) D3S6 1 6 11 16 21 26 human Nav1.1 MYLYF VIFIIFGSFF TLNLF IGVII DNF (SEQ ID NO.: 19) Nav1.2 MYLYF VIFII FGSFF TLNLFIGVII DNF (SEQ ID NO.: 20) Nav1.3 MYLYF VIFII FGSFF TLNLF IGVII DNF (SEQID NO.: 21) Nav1.4 MYLYF VIFII FGSFF TLNLF IGVII DNF (SEQ ID NO.: 22)Nav1.5 MYIYF VIFII FGSFF TLNLF IGVII DNF (SEQ ID NO.: 23) Nav1.8 MYLYFVIFII GGSFF TLNLF VGVII DNF (SEQ ID NO.: 24) rat Nav1.4 MYLYF VIFIIFGSFF TLNLF IGVII DNF (SEQ ID NO.: 25) Nav1.5 MYIYF VVFII FGSFF TLNLFIGVII DNF (SEQ ID NO.: 26) Nav1.7 MYLYF VVFII FGSFF TLNLF IGVII DNF (SEQID NO.: 27) Nav1.8 MYIYF VVFII FGGFF TLNLF VGVII DNF (SEQ ID NO.: 28)D4S6 1 6 11 16 21 26 human Nav1.1 GIFFF VSYII ISFLV VVNMY IAVIL ENF (SEQID NO.: 29) Nav1.2 GIFFF VSYII ISFLV VVNMY IAVIL ENF (SEQ ID NO.: 30)Nav1.3 GIFFF VSYII ISFLV VVNMY IAVIL ENF (SEQ ID NO.: 31) Nav1.4 GICFFCSYII ISFLI VVNMY IAIIL ENF (SEQ ID NO.: 32) Nav1.5 GILFF TTYII ISFLIVVNMY IAIIL ENF (SEQ ID NO.: 33) rat Nav1.4 GICFF CSYII ISFLI VVNMYIAIIL ENF (SEQ ID NO.: 34) Nav1.5 GILFF TTYII ISFLI VVNMY IAIIL ENF (SEQID NO.: 35)

[0007] Except for a single I→V change at position 7 of D3S6, the rat andhuman Nav1.4 and Nav1.5 sequences are identical for all four S6segments. Because of the very high degree of conservation (in factidentity) of the 11 amino acids at the carboxy termini of the S6segments, the person of skill in the art expects that substitution inthis region will have the same effect on sodium channel function acrossmammalian species and across isoforms of the Nav1 protein.

[0008] The numbering shown in the charts above is the standard numberingused to identify the 28 amino acids in the S6 segments by their positionwithin that segment. A separate system of numbering that may be appliedto those same amino acids derives from their position within thesequence of the whole protein. Because the amino acid sequences ofmembers of the Nav family of proteins vary widely outside thetransmembrane regions, the protein sequence residue numbers assigned tothe corresponding amino acids in the S6 segments differs among speciesand among sodium channel protein isoforms within species. Thus, theleucine identified as residue 19 in segment 6 in domain 1 (D1 S6) isL407 in human Nav1.5, L408 in rat Nav1.5, L441 in human Nav1.4 and L435in rat Nav1.4. Similarly, the isoleucine identified as residue 23 insegment 6 in domain 4 (D4S6) is I1770 in human Nav1.5, I1771 in ratNav1.5, I1581 in human Nav1.4 and I1589 in rat Nav1.4. Unless otherwisenoted, amino acids will be identified hereinafter, when referring to thewhole protein, according to their position in rNav1.4. Thus A438 refersto the alanine that occurs at position 438 in rNav1.4.

SUMMARY OF THE INVENTION

[0009] In one aspect the invention relates to a method or screen forassessing the potential of a compound to treat a pathological condition,such as arrhythmia, which is manifested by an increased late sodiumcurrent in a heart. The method comprises:

[0010] (a) providing a recombinant cell that expresses a mutant Nav 1sodium channel protein;

[0011] (b) measuring a first plateau current in the cell;

[0012] (c) exposing the cell to a test compound;

[0013] (d) measuring a second plateau current in the cell; and

[0014] (e) comparing the first and second currents. A lower secondcurrent indicates that the test compound is a potential anti-arrhythmicagent. The mutant sodium channel protein has an amino acid sequence inwhich one or more amino acids among the ten amino acids occurring at thecarboxy end of the S6 segments of D1, D2, D3 or D4 domains of mammalianNav1 differs from the amino acid in wild-type Nav1 by substitution withtryptophan, phenylalanine, tyrosine or cysteine. Mammalian Nav 1proteins encompassed by the present invention encompass mammalianNav1.1-Nav 1.9.

[0015] In another embodiment, the mutant sodium channel protein has anamino acid sequence in which one or more amino acids among the ten aminoacids occurring at the carboxy end of the S6 segments of D1, D2, D3 orD4 domains of mammalian Nav1.4 or Nav 1.5 differs from the amino acid inwild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosineor cysteine. In another embodiment, the mutant sodium channel proteinhas an amino acid sequence in which at least one of amino acids 19, 21and 22 of the S6 segment of D1 and amino acids 23 and 24 of the S6segment of the D4 domain of mammalian Nav1 differs from the amino acidin wild-type Nav1 by substitution with tryptophan, phenylalanine,tyrosine or cysteine.

[0016] In another embodiment, the mutant sodium channel protein has anamino acid sequence in which at least one of amino acids 19, 21 and 22of the S6 segment of D1 and amino acids 23 and 24 of the S6 segment ofthe D4 domain of mammalian Nav1.4 or Nav1.5 differs from the amino acidin wild-type Nav1.4 or Nav1.5 by substitution with tryptophan,phenylalanine, tyrosine or cysteine.

[0017] In another embodiment, the mutant sodium channel protein has anamino acid sequence in which at least one of amino acids L435, L437,A438, I1589 and I1590 of wild-type rNav1.4 is replaced by tryptophan,phenylalanine or tyrosine. L437 of rNav1.4 may be replaced by cysteine,in addition to tryptophan, phenylalanine or tyrosine.

[0018] In another aspect, the invention relates to an isolated nucleicacid comprising a nucleotide sequence that codes for a mutant mammalianNav 1 protein. The mutant protein has a sequence as described above.

[0019] In another aspect, the invention relates to a cell transfectedwith a nucleic acid that encodes a mutant mammalian Nav1 protein. Themutant protein has a sequence as described above.

[0020] In another aspect, the invention relates to a functionalrecombinant sodium channel protein containing an amino acid sequencechosen from: WILAVVAMAY SEQ ID NO.: 36 YILAVVAMAY SEQ ID NO.: 37FILAVVAMAY SEQ ID NO.: 38 LILWVVAMAY SEQ ID NO.: 39 LILYVVAMAY SEQ IDNO.: 40 LILFVVAMAY SEQ ID NO.: 41 LICWVVAMAY SEQ ID NO.: 42 LICYVVAMAYSEQ ID NO.: 43 LICFVVAMAY SEQ ID NO.: 44 WICWVVAMAY SEQ ID NO.: 45YICYVVAMAY SEQ ID NO.: 46 FICFVVAMAY SEQ ID NO.: 47 WICYVVAMAY SEQ IDNO.: 48 WICFVVAMAY SEQ ID NO.: 49 YICWVVAMAY SEQ ID NO.: 50 FICWVVAMAYSEQ ID NO.: 51 YICYVVAMAY SEQ ID NO.: 52 FICFVVAMAY SEQ ID NO.: 53YICFVVAMAY SEQ ID NO.: 54 FICYVVAMAY SEQ ID NO.: 55 LIWAVWAMAY SEQ IDNO.: 56 LIYAVWAMAY SEQ ID NO.: 57 LIFAVWAMAY SEQ ID NO.: 58 LILAVWAMAYSEQ ID NO.: 59 MYIAWILENF SEQ ID NO.: 60 MYIAYILENF SEQ ID NO.: 61MYIAFILENF SEQ ID NO.: 62 MYIAIWLENF SEQ ID NO.: 63 MYIAIYLENF SEQ IDNO.: 64 MYIAIFLENF SEQ ID NO.: 65 MYIACILENF SEQ ID NO.: 66 MYIAICLENFSEQ ID NO.: 67 MYIAWWLENF SEQ ID NO.: 68 MYIAYYLENF SEQ ID NO.: 69MYIAFFLENF SEQ ID NO.: 70

[0021] In another aspect, the invention relates to a functionalrecombinant sodium channel protein containing two sequences of aminoacids. The first amino acid sequence is chosen from: WILAVVAMAY (SEQ IDNO.: 36); LILWVVAMAY (SEQ ID NO.: 39); LICWVVAMAY (SEQ ID NO.: 42);WICWVVAMAY (SEQ ID NO.: 45), and

[0022] LILAVWAMAY (SEQ ID NO.: 59). The second amino acid sequencechosen from: MYIAWILENF (SEQ ID NO.: 60); MYIAIWLENF (SEQ ID NO.: 63);MYIACILENF (SEQ ID NO.: 66); MYIAICLENF (SEQ ID NO.: 67), and MYIAWWLENF(SEQ ID NO.: 68).

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a schematic representation of the rNav 1.4 Na⁺ channelprotein in a cell membrane.

[0024]FIG. 2 illustrates the activation of wild-type and rNav 1.4-A438Wco-expressed with β1. Families of Na⁺ currents for wild type (A) and Nav1.4-A438 mutant (B) were evoked by 5 ms pulses from the holdingpotential (−140 mV) to voltages ranging from −120 to +50 mV in 10-mVincrements. The current traces evoked by a pulse to −50 mV and to +50 mVare labeled. Normalized membrane conductance (g_(m)) (C) was determinedfrom the equation g_(m)=I_(Na)/(E_(m)−E_(Na)), where I_(Na) is the peakcurrent, E_(m) is the amplitude of the pulse voltage, and E_(Na) is thereversal potential, and plotted against the pulse voltage. Plots werefitted with a Boltzmann function, which yielded the midpoint voltage(V_(0.5)) and slope (k) for wild-type (open circles, n=5) of −32.0±0.9mV and 8.7±0.8 mV, respectively, and −38.8±1.2 mV and 13.1±11.1 mV forrNav1.5-A438W (closed circles, n=6).

[0025]FIG. 3 is a schematic representation summarizing the effects of W-and selected C- mutations at C-termini of D1 S6 and D4S6 on activationand inactivation gating.

[0026]FIG. 4 is a bar graph depicting the relative maintained currentsin various W- and C-mutations at C-termini of D1 S6 and D4S6. Fractionof non-inactivating current for D1 S6 mutants (left), D4S6 (middle), anddouble and triple mutants (right). Cells were cotransfected with β1subunit; non-expressing mutants are noted with an X. The fraction ofnon-inactivating current was determined as the averaged currentamplitude near 5 ms after the +50 mV pulse (e.g., FIG. 2) divided by thepeak current. Error bars indicate standard error. Asterisks indicatesignificant differences from the wild-type channels as determined by a ttest (p<0.05, n=4-6). Dotted line indicates the value of wild-type. Barsin gray indicate single C-substitution.

[0027]FIG. 5 shows the steady-state inactivation of wild-type andrNav1.4-A438W coexpressed with β1. Superimposed Na⁺ currents of wildtype (A) and rNav1.4-A438W mutant (B) were evoked by a 5-ms test pulseto +30 mV. Test pulses were preceded by 100-ms conditioning pulsesranging from −160 mV and −15 mV in 5-mV increments. Notice that smallnon-inactivating inward currents appeared at conditioning voltages >−60mV. Cells were cotransfected with β 1 subunit. (C) Normalized Na⁺current availability (h_(∞)) of wild-type (open circle, n=5) andrNav1.4-A438W (closed circle, n=5) were plotted as a function of the100-ms conditioning pulse voltage. Plots were fitted with the Boltzmannfunction. The fitted midpoint (h_(0.5)) and slope factor (k) forwild-type were −73.4 ±0.1 mV and 5.0±0.1 mV, respectively, and −89.0±0.3mV and 6.0±0.2 mV for Nav2.4-A438W, respectively.

[0028]FIG. 6 shows gating properties of rNav1.4-I1589W coexpressed withp 1. (A) Superimposed Na⁺ current traces were evoked by 5-ms pulses fromthe holding potential (−140 mV) to voltages ranging from −120 to +50 mVin 10-mV increments. (B) Superimposed currents were evoked by a 5-mstest pulse to +30 mV preceded by 100-ms conditioning pulses ranging from−160 mV to −15 mV in 5-mV increments. (C) Normalized Na⁺ conductance(g_(m)) was derived from (A) as described in the FIG. 2 legend, plottedagainst voltage, and fitted with a Boltzmann function. The fittedmidpoint voltage (V_(0.5)) and slope (k) of the function for wild-type(open circles, n=5) were −32.0±0.9 and 8.7±0.8, respectively, and−19.0±1.0 and 12.6±0.9, respectively for rNav1.4-I1589W (closed circles,n=6). Normalized Na⁺ current availability (h_(∞)) of wild-type (opendown triangle, n=5) and Nav1.4-I1589W (closed down triangle, n=5) werederived from (B) as described in the FIG. 5 legend, and plotted as afunction of conditioning voltage. Plots were fitted with the Boltzmannfunction. The midpoint (h_(0.5)) and slope factor (k) for wild-type were−73.4±0.1 and 5.0±0.1, respectively, and −66.5±0.2 and 5.7±0.2,respectively, for the Nav1.4-I1589W. Cells were cotransfected with β1subunit.

[0029]FIG. 7 shows activation of rNav1.4-A438C and rNav1.4-I1589Ccoexpressed with β1. Superimposed Na⁺ current families of Nav1.4-A438C(A) and Nav1.4-I1589C (B) were evoked by 5-ms pulses from holdingpotential of −140 mV to voltages ranging from −120 to +50 mV in 10-mVincrements. (C) Normalized membrane conductance (g_(m)) plotted versusthe amplitude of the 5-ms voltage step. Gm was determined as describedin the FIG. 2 legend, plotted against the membrane voltage, and fittedwith a Boltzmann function. The fitted midpoint voltage (V_(0.5)) andslope (k) of the function for wild-type (open circles, n=5) were−32.0±0.9 and 8.7±0.8, respectively, −25.9±0.7 and 8.3±0.6 forNav1.4-A438C (closed square, n=5), and −39.9±1.1 and 10.9±0.9 forNav1.4-I1589C (closed triangle, n=6). Cells were cotransfected with β1subunit.

[0030]FIG. 8 shows activation gating of double and triple mutants.Superimposed Na⁺ current families were evoked by 5-ms pulses fromholding potential of −140 mV to voltages ranging from −120 to +50 mV in10-mV increments for mutants A438W/I1589W (A), L437C/A438W (B),L435W/L437C/A438W (C), and I1589W/I1590W (D). All mutants wereco-transfected with the β1 subunit.

[0031]FIG. 9. shows slow inactivation gating of double and triplemutants. To induce slow inactivation, we applied conditioning prepulsesranging from −180 mV to 0 mV with a duration of 10 s. After a 100-msinterval at −140 mV, Na⁺ currents were evoked by the delivery of a +30mV test pulse. (A) Peak Na⁺ Currents were normalized to thecorresponding current obtained with a prepulse to −180 mV and plottedagainst conditioning prepulse potential. Data were fitted with aBoltzmann function. The fitted V_(0.5) values and k (slope factor)values from the Boltzmann functions were (in mV)-51.1±0.5 and 9.7±0.4,respectively, for wild-type (open circle, n=5); -49.9±0.1 and 5.0±0.1,respectively, for L435W/L437C/A438W (closed circle, n=5); -45.2±0.3 and5.8+0.2, respectively, for L437C/A438W (closed up triangle, n=5);-45.2±0.3 and 6.8±0.3, respectively, for A438W/I1589W (closed downtriangle, n=6); and −50.0±0.9 and 9.5±0.8, respectively, forI1589W/I1590W (closed diamond, n=5). The final steady-statenon-inactivated values (in %) were 57.5±0.4 (wild-type), 3.3±0.4(L435W/L437C/A438W), 12.3±0.6 (L437C/A438W), 11.6±0.8 (A438W/I1589W),and 3.4±1.7 (I1589W/I1590W). All mutants are co-transfected with the β1subunit. (B) Development of slow inactivation. For the development ofslow inactivation, the prepulse duration at +30 mV was varied rangingfrom 0 to 10s. The peak current at the test pulse of +30 was measuredand normalized to the initial peak amplitude without a prepulse, andthen plotted against the prepulse duration. The data were fitted by asingle-exponential function. The τ values (and final steady-state Y₀values) for wild-type, L435W/L437C/A438W, L437C/A438W, A438W/I1589W, andI1589W/I1590W are 4.8±0.3 s (51.6%), 0.72±0.01 s (3.0%), 0.50±0.01 s(16.6%), 0.64±0.02 s (10.5%), and 0.81±0.02 s (16.6%), respectively.

[0032]FIG. 10 shows a coarse correlation between fast and slowinactivation gating. (A) The relative level of slow inactivation for D1S6 mutants (left), D4S6 (middle), and double and triple mutants (right).Non-expressing mutants are noted with an X. Values represent mean±S.E.of peak current elicited by a 5 ms test pulse to +30 mV preceded by a10-s conditioning pulse to 0 mV and a 100 ms interval at −140 mV asdescribed in FIG. 9A. Asterisks indicate significant differences fromthe wild-type channels as determined by a t test (p<0.05). Dotted lineindicates the value of wild-type. Bars in gray indicate singleC-substitution. (B) The fraction of slow-inactivated current vs. thefraction of non-inactivating current of the individual mutant. Data forwild type and mutants were taken from FIG. 4 for x axis and from FIG.10A for y axis. The mutants are labeled. The solid line is the linearfit of the complete data set with a correlation coefficient (r) of 0.61.Mutants with impaired fast inactivation (fraction of non-inactivatingcurrent >5%) are shown at the right-hand side of the dashed line; all ofthem show enhanced slow inactivation.

[0033]FIG. 11 depicts voltage dependence of flecainide block in rNav1.4channels. Conditioning prepulses ranging from −180 mV to −10 mV wereapplied for 10s. After a 100-ms interval at −140 mV, Na⁺ currents wereevoked by a test pulse at +30 mV for 5-ms. Currents recorded before(open circle, n=6) and after 30 μM flecainide (closed circle, n=6) werenormalized to the current obtained at the −180 mV conditioning pulse andplotted against the conditioning voltages. Flecainide data were thenrenormalized at each voltage with respect to the control value withoutflecainide. Data were fitted with a Boltzmann function(1/[1+exp((V_(0.5)−V)/k_(Ε))]). The average V_(0.5) value and k_(Ε)(slope factor) value for the fitted functions were (in mV) −47.9±1.1 and8.6±0.9, respectively, for control and −57.3±2.7 and 17.6±2.1,respectively, for flecainide.

[0034]FIG. 12 shows blockade of inactivation-deficient Nav1.4L435W/L437C/A438W channels at various flecainide concentrations.Superimposed Na⁺ currents evoked by a 140 ms test pulse to +30 mV every30 seconds were shown at various flecainide concentrations. Steady stateblock at each concentration was achieved within 5 min.

[0035]FIG. 13 shows the decay phase of the Na⁺ current; the decay phaseof the Na⁺ current was fitted with a single exponential function, andthe corresponding time constant (τ) was inverted and plotted against thecorresponding concentration. Data were fitted with a linear regressiony=14.9x+12.16 (solid line). On-rate (k_(on)) corresponded to the slopeof the fitted line (14.9 μM⁻¹s⁻¹) and the off-rate (k_(off))corresponded to the y-intercept (12.16 s-1). The dissociation constantwas determined by the equation K_(D)=k_(off)/k_(on) and equaled 0.81 μM.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The first aspect of the invention relates to a screen forassessing the potential of a compound to treat a pathological condition,such as arrhythmia, which is manifested by an increased late sodiumcurrent in a heart. The method comprises:

[0037] (a) providing a cell that expresses a recombinant mutantNav1sodium channel protein;

[0038] (b) measuring a first plateau current in the cell;

[0039] (c) exposing the cell to a test compound;

[0040] (d) measuring a second plateau current in the cell; and

[0041] (e) comparing the first and second currents. A lower secondcurrent indicates that the test compound is a potential anti-arrhythmicagent. The mutant sodium channel protein has an amino acid sequence inwhich one or more amino acids among the ten amino acids occurring at thecarboxy end of the S6 segments of D1, D2, D3 or D4 domains of amammalian Nav1 differs from the amino acid in wild-type Nav1.4 or bysubstitution with tryptophan, phenylalanine, tyrosine or cysteine. In apreferred embodiment, the mutant sodium channel protein has an aminoacid sequence in which at least one amino acid chosen from amino acids19, 21 and 22 of the S6 segment of D1 and amino acids 23 and 24 of theS6 segment of the D4 domain of a mammalian Nav1 is the amino acid thatis replaced. These amino acids correspond to amino acids L435, L437,A438, I1589 and I1590 of wild-type rNav1.4. The wild-type amino acidsmay be replaced by tryptophan, phenylalanine or tryrosine, all of whichare neutral, hydrophobic and bulky—the important common features forimpairing the so-called “fast inactivation” of the sodium channel.Certain of the wild-type amino acids may also be replaced by cysteine.Cysteine produces a similar impairment of fast inactivation, but appearsto do so by an indirect route, whereby it achieves effective bulkiness(and hydrophobicity) through reaction of the sulfhydryl withphysiologically accessible nucleophiles. The experiments described belowwere carried out with tryptophan and cysteine.

[0042] The remainder of the Nav protein—outside the S6 segments—isoptimally the sequence of a Nav 1 sodium channel protein, for example,Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, Nav 1.8or Nav 1.9. Nav1.4 and Nav1.5 are the two isoforms of the Nav proteinthat are found in skeletal and heart muscle; the remaining isoforms havebeen primarily identified in the CNS and neuronal structures. In oneembodiment, a leucine corresponding to L437 of rNav1.4 is replaced withcysteine, and one or both of a leucine and an alanine corresponding toL435 and A438 respectively are replaced with tryptophan. In otherembodiments, alanine corresponding to A438 and an isoleucinecorresponding to I1589 are replaced, preferably by tryptophan. Preferredsequences of the residues 19-28 of the S6 segment are: WILAVVAMAY SEQ IDNO.: 36 YILAVVAMAY SEQ ID NO.: 37 FILAVVAMAY SEQ ID NO.: 38 LILWVVAMAYSEQ ID NO.: 39 LILYVVAMAY SEQ ID NO.: 40 LILFVVAMAY SEQ ID NO.: 41LICWVVAMAY SEQ ID NO.: 42 LICYVVAMAY SEQ ID NO.: 43 LICFVVAMAY SEQ IDNO.: 44 WICWVVAMAY SEQ ID NO.: 45 YICYVVAMAY SEQ ID NO.: 46 FICFVVAMAYSEQ ID NO.: 47 WICYVVAMAY SEQ ID NO.: 48 WICFVVAMAY SEQ ID NO.: 49YICWVVAMAY SEQ ID NO.: 50 FICWVVAMAY SEQ ID NO.: 51 YICYVVAMAY SEQ IDNO.: 52 FICFVVAMAY SEQ ID NO.: 53 YICFVVAMAY SEQ ID NO.: 54 FICYVVAMAYSEQ ID NO.: 55 LIWAVWAMAY SEQ ID NO.: 56 LIYAVWAMAY SEQ ID NO.: 57LIFAVWAMAY SEQ ID NO.: 58 LILAVWAMAY SEQ ID NO.: 59 MYIAWILENF SEQ IDNO.: 60 MYIAYILENF SEQ ID NO.: 61 MYIAFILENF SEQ ID NO.: 62 MYIAIWLENFSEQ ID NO.: 63 MYIAIYLENF SEQ ID NO.: 64 MYIAIFLENF SEQ ID NO.: 65MYIACILENF SEQ ID NO.: 66 MYIAICLENF SEQ ID NO.: 67 MYIAWWLENF SEQ IDNO.: 68 MYIAYYLENF SEQ ID NO.: 69 MYIAFFLENF SEQ ID NO.: 70

[0043] The resultant protein may also contain a second amino acidsequence, YMIFFX^(a)X^(b)X^(c)IFLGSFYLX^(d)N (SEQ ID NO. 71),amino-terminal to the foregoing amino acid sequence. In the secondsequence, X^(a) is V or M; X^(b) is L or V; and X^(c) and X^(d) areindependently I or V. These variable residues account for the variantsthus far observed in S6 residues 1-18 of mammalian Nav proteins. Forexample, one S6 sequence according to the invention would beYMIFFMLVIFLGSFYLVNWILAVVAMAY (SEQ ID NO. 72).

[0044] As will be evident, functional recombinant sodium channelproteins may contain multiple sequences of amino acids altered in the S6segments of different domains. In preferred multiple-sequenceembodiments, a first amino acid sequence may be chosen from: WILAVVAMAY(SEQ ID NO. 36); LILWVVAMAY (SEQ ID NO. 37); LICWVVAMAY (SEQ ID NO. 42);WICWVVAMAY (SEQ ID NO. 45), and LILAVWAMAY (SEQ ID NO. 59), and a secondamino acid sequence chosen from: MYIAWILENF (SEQ ID NO.

[0045] 60); MYIAIWLENF (SEQ ID NO. 63); MYIACILENF (SEQ ID NO. 66);MYIAICLENF (SEQ ID NO. 67), and MYIAWWLENF (SEQ ID NO. 68). For obviousreasons, the two S6 sequences will not commonly be adjacent each otherin primary sequence. In fact, in preferred embodiments, the sequenceswill be separated by at least 400 amino acid residues, and, when thesequences reflect S6 segments in domains 1 and 4, they will be separatedby at least 1000 residues.

[0046] In another aspect, the invention relates to a cell comprising arecombinant nucleic acid that encodes a mutant mammalian Nav1 protein.The mutant protein has a sequence as described above, and the cellexpresses sodium channels that exhibit a plateau current of greater than1 nanoamp. The process for transfecting a cell with an appropriatenucleic acid is well known in the art. The particular cell chosen fortransfection was the human embryonic kidney (HEK293t) cell. Chinesehamster ovary cells are also well suited for this purpose. The person ofskill will be aware of cell lines that become known in the art for theirability to express proteins of the size (200 kDa) of the Nav protein,and these will be appropriate for transfection with the nucleic acids ofthe invention. Any eukaryotic cells which support stable replication ofplasmids containing nucleic acids encoding the mutant sodium channelsdescribed above may be used in practicing the invention. Non-limitingexamples of host cells for use in the present invention include HEK 293cells (American Type Culture Collection, Manassas, Va. (ATCC Cat. No.CRL-1573), CV-1/EBNA-1 cells (ATCC Cat. No. CRL 10478), HeLa cells,D98/raji cells, 293EBNA (Invitrogen, Cat. No. R62007), CV-1 cells (ATCCCat. No. CCL 70) and 143B cells (ATCC Cat. No. CRL-8303). In addition,primary cultures of eukaryotic cells may be isolated from their tissueof origin and transfected with the appropriate expression vector.

[0047] One of the advantages of the substitutions of the presentinvention is that, unlike all previously reported mutants of the Navprotein, substitution with W, F, Y or C in the carboxy terminal tenresidues of S6, when expressed at a useful level, results in a cell linethat is viable (the leakage is not lethal to the cell), while at thesame time the cells exhibit a large enough sodium channel current tomake measurement reliable.

[0048] In practicing the present invention, many conventional techniquesin molecular biology, microbiology, and recombinant DNA are used. Suchtechniques are well known and are explained in, for example, Sambrook etal., 2001, Molecular Cloning: A Laboratory Manual, Third Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: APractical Approach, Volumes I and II, 1985 (D. N. Glover ed.);Oligonucleotide Synthesis, 1984 (M. L. Gait ed.); Nucleic AcidHybridization, 1985, (Hames and Higgins, eds.); Transcription andTranslation, 1984 (Hames and Higgins, eds.); Animal Cell Culture, 1986(R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986, (IRL Press);Perbas, 1984, A Practical Guide to Molecular Cloning; the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wuand Grossman, and Wu, eds., respectively); Current Protocols inMolecular Biology, John Wiley & Sons, Inc. (1994), and all more currenteditions of these publications. Throughout this application, variousreferences are referred to within parentheses or square brackets. Thedisclosures of these publications in their entireties are herebyincorporated by reference as if written herein.

[0049] In the description that follows, certain conventions will befollowed as regards the usage of terminology. The term “expression”refers to the transcription and translation of a structural gene (codingsequence) so that a sodium channel protein (i.e. expression product)having biological activity is synthesized. It is understood thatpost-translational modifications may remove portions of the polypeptidethat are not essential and that glycosylation and otherpost-translational modifications may also occur. The term“transfection,” as used herein, refers to the uptake, incorporation andexpression of exogenous DNA into a host cell by any means, and includes,without limitation, transfection of plasmids, episomes and othercircular DNA forms. The expression vector may be introduced into hostcells via any one of a number of techniques including but not limited toviral infection, transformation, transfection, lipofection or othercationic lipid based transfection, calcium phosphate co-precipitation,gene gun transfection, and electroporation. These techniques are wellknown to persons of skill in the art.

[0050] The term “sodium channel protein” refers to any protein thatprovides a functional sodium channel in an excitable membrane. Knownsodium channel proteins are the isoforms of the Nav family: Nav1.1through Nav 1.9, Nav 2.1 through Nav2.3 and Nav 3.1. [See GoldinAnn.N.Y.Acad.Sci 868:38-50 (1999)]. For the present invention, type INav proteins (referred hereinafter as Nav 1 or Nav 1.x) are preferred,with Nav1.4 and Nav 1.5 being more preferred. The term “mutant sodiumchannel protein” or “recombinant sodium channel protein” refers to arecombinant protein having the sequence of a Nav 1 protein, that is, Nav1.1 through Nav 1.9, in which from one to ten amino acids differ fromthe wild-type. The person of skill will of course recognize that inproteins of 2000 amino acids, such as those of the Nav family, there canbe innumerable deletions, insertions and substitutions that do notaffect the function of the protein in any measurable way. Proteinshaving >90% homology to a protein in the Nav family but containingdeletions, insertions and substitutions that do not affect theirfunction in providing a sodium channel are to be considered equivalentsof the claimed mutants. Furthermore, because the genetic code isdegenerate, more than one codon may be used to encode a particular aminoacid, and therefore, the amino acid sequence can be encoded by any setof similar DNA oligonucleotides. With respect to nucleotides, therefore,the invention encompasses all the DNA sequences containing alternativecodons, which code for the eventual translation of the identical aminoacid.

[0051] The term “plateau current” refers to the current measured in asingle cell 5 ms after activation by a sufficient voltage pulse to openthe channel. For cells expressing wild-type Nav1.4 and 1.5 the pulse is−60±10 mV and the plateau current is below 1 nA. For cells expressingmutant Nav1.4 and 1.5 according to the invention, the pulse can besomewhat higher (e.g. −70±10 mV) and the plateau current is above 1 nA.

[0052] The utility of the mutant Nav test system has been demonstratedwith flecainide. Flecainide is one of several orally active class Icantiarrhythmic drugs. The primary target of flecainide is the cardiacNa⁺ channel, which is responsible for the upstroke of cardiac actionpotentials. Recently, flecainide has been found effective for patientswith the Long QT syndrome [Windle, et al., Ann. NoninvasiveElectrocardiol. 6(2):153-158 (2001)]. The state-dependent binding offlecainide with wild type and an exemplary inactivation-deficient sodiumchannel of the invention (rNav1.4-L435W/L437C/A438W) were compared inthe HEK293t expression system. Unlike the inactivation-deficient cardiacNav1.5 IFM/QQQ mutant of Grant et al. [Biophysical J. 79:3019-3035(2000)], the channel of the invention expressed well, which is evidentfrom the large sodium currents (>1 nanoamp following −50 mVstimulation), and we demonstrate below that flecainide binds rapidly andpreferentially with the open state but minimally with the resting state.This provides the basis for the first truly useful, high-throughputscreen for agents that may be used to treat various pathologicalconditions that manifest an increase in persistent late sodium currentsin the heart. Such agents include antiarrhythmic agents. To screen forsuch agents, one follows the procedure described in the experimentsdescribed below, and one simply replaces flecainide by a test agent.

[0053] The invention began from the hypothesis that an amino acid havinga bulky hydrophobic side chain, would disrupt or alter channel functionbecause of its large size. The disruption or alteration would occur if alarge hydrophobic amino acid were substituted for an amino acid thatcontacts or directly interacts with other parts of the channel protein.In addition, it was possible that the effects of several residues on thefast inactivation gating would be additive after multiple substitutions.The experiments below employed tryptophan (W) and cysteine (C) as theprototypic bulky, hydrophobic amino acids.

[0054] In practicing the method of the invention, a mammalian mutant Nav1 sodium channel protein is expressed in an appropriate cell. The cellexpressing the sodium channel of the invention is contacted with acompound to determine whether the compound has potential utility as ananti-arrhythmic agent. Isolated nucleic acids comprising a nucleotidesequence that codes for a mutant mammalian Nav 1 protein according tothe invention may be obtained by methods known to one of skill in theart. Site-directed mutagenesis of DNA from appropriate cells, forexample, heart and smooth muscle, or cell line cultures of theappropriate species or tissue, is then performed to obtain a nucleicacid encoding mutant sodium channel protein as described above.

[0055] Isolation of DNA

[0056] DNA encoding a Na⁺ channel, in accordance with the instantinvention, may be obtained by screening reverse transcripts of mRNA orcDNA from appropriate cells or tissues, for example, CNS, skeletalmuscle, denervated skeletal muscle, cardiac muscle, uterus, astrocytesor cell line cultures of the appropriate tissues, by screening genomiclibraries, or by combinations of these procedures. Screening of mRNA,cDNA or genomic DNA may be carried out with oligonucleotide probesgenerated from the nucleic acid sequence information of the sodiumchannel proteins disclosed herein.

[0057] An alternative means to isolate the gene encoding a Nav sodiumchannel protein is to use polymerase chain reaction (PCR) methodology asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.

[0058] Site-Directed Mutagenesis

[0059] The QUIKCHANGE XL™ site-directed mutagenesis kit (Stratagene, LaJolla, Calif.) was used to create rat skeletal muscle Nav1.4 mutantclones as previously described (Wang and Wang, Biophys. J. 72:1633-1640,1997; Wang and Malcolm, BioTechniques 26:680-682, 1999). Preliminarily,a wild type rNav1.4-pcDNA1/Amp clone was generated to serve as thetemplate for mutagenesis. Briefly, a cDNA insert prepared from the wildtype rat muscle cDNA Nav 1.4, clone μ1-2, (Genbank accession numberM26643) (Trimmer et al., Neuron 3: 33-49, 1989) was cloned into theEcoRI site of a pcDNA1/Amp vector (Invitrogen, Carlsbad, Calif.) toyield the vector rNav 1.4-pcDNA1/Amp. For mutagenesis, two complementarymutant oligonucleotides of 38-42 nucleotides in length (see Table 1) areannealed to the template DNA in separate tubes for 4 cycles of PCRreaction (94° C., 30 sec; 55° C., 1 min, 68° C., 23 min). TABLE 1Primers for Site-Directed Mutagenesis Clones rNav 1.4 437C438W/rNav 1.4435W437C438W: 5′- ctcatcaatctgatctgctgggtggtggccatggcgtac - 3′ (SEQ IDNO.: 73) 5′- cctcatcaattggatctgctgggtggtggccatggcgtac- 3′ (SEQ ID NO.:74) Clones hNav 1.4 443C444W/hNav 1.4 441W443C444W 5′-cctcatcaatctgatctgctgggtggtggccatggcatatg - 3′ (SEQ ID NO.: 75) 5′-gctctttctacctcatcaattggatctgctgggtggtggccatggcatatgc - 3′ (SEQ ID NO.:76) hNav 1.5 409C410W 5′- cctggtgaacctgatctgctgggtggtcgcaatggcc - 3′(SEQ ID NO.: 77) 5′- ccttctacctggtgaactggatctgctggg - 3′ (SEQ ID NO.:78)

[0060] The PCR reaction mix contains template DNA (0.4 ng/ul), primer(5ng/ul), KCl (10 mM), (NH 4)₂SO₄(10 mM), Tris-HCl (pH8.8) (20 mM),MgSO₄ (2 mM), TritonX-100 (0.1%), 0.1 mg/ml bovine serum albumin,deoxynucleotides mix (0.4 mM each), pfuTurbo DNA polymerase (0.05 U/ul).The stage 2 PCR reactions follow after mixing the two primer stage 1reactions into one and perform the following PCR reactions: 94° C., 30sec, 18 cycles of (94° C., 30sec, 55° C., 1 min; 68° C., 23 min). The invitro synthesized DNA is digested with Dpnl at 0.2 U/ul at 37° C. forone hour. One μl of the DpnI treated DNA is transformed into XL-Goldultracompetent cells (Strategene, La Jolla Calif.), plated on Ampicillin(50 ug/ml) LB plates. Bacterial colonies are picked into LB containingAmpicillin at 50 ug/mlDNA. The mutation frequency is 25-100%, that is,you will obtain at least one mutant if you sequence 4 clones.

[0061] To minimize the possibility that unique phenotypes are due tounwanted mutations, independent clones of rNav1.4-L435W/L437C/A438W andrNav1.4-L437C/A438W as well as additional homologous L435W/L437C/A438Wclones from human isoforms (hNav1.4 and hNav1.5) were created.Preliminary results showed that all of these independent and homologousclones displayed comparable phenotypes to those of rNav1.4 counterparts.DNA sequencing near the mutated site was performed to confirm themutations.

[0062] Transient Transfection

[0063] Transfection methods are well known in the art. In oneembodiment, human embryonic kidney (HEK293t) cells were grown to ˜50%confluence in DMEM (Gibco) containing 10% fetal bovine serum (HyClone,Logan Utah), 1% penicillin and streptomycin solution (Sigma, St. Louis,Mo.), 3 mM taurine, and 25 mM HEPES (Gibco). HEK293t cells were thentransfected with cloned Na⁺ channels, either wild type or mutant, by acalcium phosphate precipitation method in a TI-25 flask (Cannon andStrittmatter, 1993). A reporter plasmid CD8-pih3m and cDNA clone in thepcDNA1/amp vector (Invitrogen, San Diego, Calif.) were prepared in 250mM CaCl₂, added to a test tube containing 0.36 ml of Hanks' balancedsalt solution and incubated at 22° C. for 20 min. The DNA solution wasthen dripped over a cell culture (30-50% confluence) containing 7 ml ofDMEM. The transfected cells were trypsinized and replated 15 h later toan appropriate density in 35-mm tissue culture dishes containing 2 ml offresh DMEM. Transfected cells were maintained at 37° C. in a humidified5% CO₂ incubator, and used after 1-4 days. Transfection-positive cells,which were identified by binding to immunobeads (CD8-Dynabeads, Dynal,Lake Success N.Y.) coated with a monoclonal antibody specific for CD8antigen, were selected for patch-clamp experiments.

[0064] Transfection of wild type rNav1.4-pcDNA1/Amp or mutant clones(5-10 μg) along with β1-pcDNA 1/Amp (10-201g) and reporter CD8-pih3m (1μg) generated sufficient sodium channel expression for later currentrecording.

[0065] Stable Transfection

[0066] For stable transfection, a selectable marker plasmid is includedin the expression vector. Generally, a drug resistance gene, forexample, pac, which confers resistance to the antibiotic puromycin isused. After transfection, the cells are trypsinized, transferred intolarge culture dishes and treated with the antibiotic for whichresistance has been conferred by the plasmid. A drug killing curveshould be determined for each batch of drug solution and cells. Two tothree weeks after transfection, drug resistant colonies are picked,amplified, and analyzed for channel expression. Other types ofselectable markers include, for example, geneticin (Invitrogen,Carlsban, Calif.), and hygromycin and can be used to obtain permanentcell lines for sodium channel expression.

[0067] Measurement of Na⁺ Current

[0068] Whole-cell configuration was used to record Na⁺ currentsaccording to the method of Hamill et al. [Pflugers Arch. 391:85-100(1981)]. Borosilicate micropipettes (Drummond Scientific Company,Broomall, Pa.) were pulled with a puller (P-87, Sutter InstrumentCompany, Novato, Calif.) and heat polished. Pipette electrodes contained100 mM NaF, 30 mM NaCl, 10 mM EGTA, and 10 mM HEPES adjusted to pH 7.2with CsOH. The pipette electrodes had a tip resistance of 0.5 to 1.0 MΩ.Access resistance was 1-2 MΩ and was further reduced by seriesresistance compensation. All experiments were performed at roomtemperature (22-24° C.) under a Na⁺-containing bath solution with 65 mMNaCl, 85 mM choline Cl, 2 mM CaCl₂, and 10 mM HEPES adjusted to pH 7.4with tetramethylammonium hydroxide. Residual outward currents wereevident in some cells at voltages >+30 mV; these currents were presentin untransfected cells and were insensitive to tetrodotoxin. Theseresidual currents were not subtracted from the measurements. Whole-cellcurrents were measured by an AXOPATCH 200B™ (Axon Instruments, FosterCity, Calif.) or an EPC-7 (List Electronics, Darmstadt/Eberstadt,Germany), filtered at 3 kHz, collected, and analyzed with pClamp8software (Axon Instruments). Leak and capacitance were subtracted by thepatch clamp device and further by the leak subtraction protocol (P/-4).Cells were held at −140 mV for functional characterizations. Voltageerror was <4 mV after series resistance compensation. An unpairedStudent's t test was used to evaluate estimated parameters (mean±SEM orfitted value±SE of the fit); P values of <0.05 were consideredstatistically significant.

[0069] Gating properties of W substitutions within the C-terminus of D1S6 in Nav1.4 Na⁺ channels were examined. To characterize the effects ofW substitutions, we measured Na⁺ currents of D1 S6 W-substituted mutantchannels at various voltages. Each of residues 19 to 28 (the carboxyterminus of S6) was replaced by W. As an example, FIGS. 2A and B showthe superimposed current families of Nav1.4 wild type and mutantNav1.4-A438W (position 22 at D1 S6) cotransfected with β1 subunit,respectively. Activation threshold was around −50 mV for wild type andaround −60 mV for A438W mutant channels. The peak conductance wascalculated as described in the figure legend, normalized, and plottedagainst the corresponding voltage (FIG. 2C). Voltage dependence ofactivation was fitted by a standard Boltzmann equation and the W mutantshowed an apparent leftward shift of −−6.8±2.1 mV (n=6). FIG. 3 is asummary of effects of W- and selected C-mutations at C-termini of D1S6and D4S6 on activation and inactivation gating. In the left panel is avertical representation of amino acid sequences of D1 S6 (top), D4S6(middle), and double and triple mutants (bottom). All mutants andwild-type Na⁺ channels were co-transfected with the β1 subunit.Activation Shift: the bar graph shows the differences in voltage for thehalf-maximal activation (V_(0.5)) of the wild-type and mutant Na⁺channels. The V_(0.5) values (mean±SEM) were obtained from the Boltzmannfits of normalized conductance versus voltage plots as described abovefor FIG. 2. Significantly, the estimated reversal potential (E_(Na))remained about the same in these mutants. Bars in gray indicate singleC-substitution. Non-expressing mutants are noted with an X. Slope Effect(Activation, Inactivation): this bar graph shows the differences in kvalues for the wild-type and mutant channels. The k values (mean±SEM)were obtained from Boltzmann fits of I/V plots (activation) and fromBoltzmann fits of steady-state inactivation plots (inactivation), asdescribed for FIG. 2 and FIG. 5, respectively. Inactivation Shift: thebar graph shows the differences in voltage for the half-maximalinactivation (h_(0.5)) of the wild-type and mutant Na⁺ channels. Theh_(0.5) values (mean±SEM) were obtained as described for FIG. 5. Allvalues were derived from n=4-6, except E1592W with n=3. An asterisk (*)indicates that the value is statistically different from that of thewild type (p<0.05). Except for L435W and 1436W, all other D1S6 mutants Wdisplayed a leftward shift. L437W shifted leftward by as much as−22.1±2.0 mV (n=5). The slope factor for each W mutant showed either nosignificant change or became less steep.

[0070] Another noticeable change in gating after A438W substitution wasthe non-inactivating currents maintained at the end of the pulse (FIG.2B vs. 2A). To quantify this difference between wild type and mutantchannels we measured the amount of the non-inactivating maintainedcurrent near the end of 5-ms+50 mV pulse. The wild-type current decayedrapidly and reached its steady-state level within 2-3 ms and 2.9±0.8%(n=5) of currents were maintained under these experimental conditions.In contrast, Nav1.4-A438W mutant showed conspicuous maintained currentsunder identical conditions. As measured near the 5-ms time, 31.9±4.2%(n=6) of currents were non-inactivating. Thus, a substitution of W atposition 22 of D1 S6 impairs the Na⁺ channel fast inactivationsignificantly (P<0.001). The relative non-inactivating components of allmutants at D1 S6 are listed in FIG. 4 (left section). The two other Wmutants with impaired fast inactivation are L435W (FIG. 4; the fractionof non-inactivating current=0.10±0.02, n=5, P<0.05; position 18) andL437W (0.055±0.017, n=5, P=0.20; position 21).

[0071] Using various conditioning pulses from −160 mV to −15 mV, wefurther characterized the steady-state inactivation of the mutantchannels. FIGS. 5A and B show Na⁺ currents of the wild type andNav1.4-A439W mutant, respectively, under these pulse conditions. Peakcurrents were measured, normalized with respect to the peak current at−160 mV and plotted against conditioning voltages (FIG. 5C). Clearly, anon-inactivating component was again present in Nav1.4-A438W mutantchannels. The data were fitted with a standard Bolzmann equation and theshift in the ΔV_(0.5) is shown in FIG. 3 (top on right side) along withthe shift in the slope factor, the Δk value.

[0072] The residues from position 19 to 26 in D4S6 were also substitutedwith tryptophan (FIG. 1; solid bracket). FIGS. 6A and B show the currentvoltage relationship and steady state inactivation measurement of mutantNav1.4-I1589W, respectively. FIG. 6C shows the normalized peakconductance and h_(∞) measurements against voltage of this mutantchannel. Again there were significant non-inactivating currentsmaintained at the end of test pulse for I1589W. The relative amounts ofthe maintained currents of all mutants at D4S6 are listed in FIG. 4(middle section) along with D1S6 mutants. The activation of I1589W wasshifted rightward by 13.0±1.9 mV (n=6) and the steady state inactivationwas shifted rightward by 6.8±0.3 mV (n=5). These changes in gatingparameters of all D4S6 mutants are listed in FIG. 3 (middle section).Two W mutant channels (I1589W and I1590W) appeared to have significantlyimpaired fast inactivation. Two mutants, I1587W and A1588W, expressedNa⁺ currents below 1 nA in this expression system.

[0073] There is not a clear relationship between the size of residue inthe native amino acid and the degree of the impairment in fastinactivation. FIGS. 7A and B show the current families of A438C andI1589C, respectively. A438W and 11590W exhibited significantly impairedfast inactivation but A438C and I1590C do not (FIGS. 2,4). In contrast,I1589C displayed impaired fast inactivation similar to that of I1589W(FIG. 4). This lack of direct correlation in volume suggests that eitherallosteric effects occur after amino acid substitutions (i.e. thesulfhydryl undergoes reaction with a nearby bulky, hydrophobicnucleophile) or these residues may specifically and directly interactwith other parts of channel structure, such as the inactivation gate.

[0074] Gating properties of double and triple substitutions of residueswithin D1S6 and D4S6 were also tested. Selected residues (L435, L437,A438, 11589, I1590) were multiply substituted. Severalmultiple-substituted mutants expressed a high level of Na⁺ currentscomparable to that of wild type. There were two distinct types ofphenotypes from these mutants. One type showed supra-additive effects onthe fast inactivation and the other showed sub-additive effects. FIGS.8A, B, C and D show the current families of A438W/1589W, L437C/A438W,L435W/L437C/A438W, and I1589W/I1590W, respectively. The results thusdemonstrate that it is feasible to create fast-inactivation deficientmutants that express well in a mammalian expression system.

[0075] When the fast inactivation was hampered by pronase or bysite-directed mutagensis, slow inactivation gating not only remainedfunctional but also was accelerated considerably. This inverserelationship suggests that the fast inactivation and slow inactivationgating have distinct identities and yet these two gating processes aresomehow coupled. To determine whether such inverse relationship holdstrue in S6 mutants with severely impaired fast inactivation, wetherefore measured the slow inactivation gating with a 10-s conditioningprepulse at various voltages. With a gap of 100 ms at −140 mV, whichallowed channels to recover from their fast inactivation but not fromtheir slow inactivation, we observed that 57.1±3.6% (n=5) of wild typeNa⁺ currents were slow inactivated at 0 mV for 10 s (FIG. 9A; opencircles). In contrast, almost all L435W/L437C/A438W mutant channels wereslow-inactivated (FIG. 9A, closed circles) at 0 mV under theseexperimental conditions. It appeared that this enhanced slowinactivation is in part due to the enhanced forward rate constant asshown in FIG. 9B. Multiple-substituted mutants with enhanced slowinactivation were inactivated with a rather rapid rate, with a timeconstant of <1 sec at +30 mV (vs. 4.8 s for wild type). It is noteworthythat slow inactivation in wild type channels does not reach its steadystate with a 10-s conditioning pulse even at +30 mV (FIG. 9B).Nonetheless, this pulse protocol allowed us to determine which mutantsexhibit altered slow inactivation significantly. In general, we observedthat mutants with the most impaired fast inactivation (L435, L437, A438,I1589, I1590) were those with enhanced slow inactivation. In particular,the multiple-substituted mutants, such as L437C/A438W andL435W/L437C/A438W, with the most impaired fast inactivation also had themost enhanced slow inactivation.

[0076] The study demonstrates that most mutants with a single Wsubstitution at the C-terminus of D1 S6 and D4S6 express observable Na⁺currents in HEK293t cells. Substitutions with W in this region alter Na⁺channel activation, fast inactivation, and/or slow inactivation gatingin varying degrees, dependent on the position of the substitution. Fivepositions (L435, L437, A438, I1589, and I1590) appear to be closelyassociated with fast inactivation gating. Two mutants (L437C/A438W andL435W/L437C/A438W) with multiple W or C substitutions exhibit minimalfast inactivation. Interestingly, all mutants with impaired fastinactivation display an enhanced slow inactivation phenotype. The orderof the level of the maintained current in D1S6/D4S6 residues is asfollows: A438W (31.9%)>I1590W (20.0%)>I1589C (18.6%)>I1589W(14.5%)>L435W (9.5%)>L437C (6.7%)>L437W (5.5%)>wild type (2.9%). Theamount of maintained current of L437W and L437C is higher than that ofthe wild type but it did not reach the level of statisticalsignificance. In comparison, Nav1.2-L421C (equivalent to Nav 1.4-L437C)has a maintained current of ˜10% of the peak current, significantlyhigher than its wild type (˜2%). Evidently, differences in the amount ofmaintained currents exist between mutants derived from differentisoforms. The magnitude of the slow inactivation and the fastinactivation appear to have an apparent inverse relationship. Thefraction of slow-inactivated channels followed the order I1590W(85.9%)>I1589W (80.6%)>A438W (76.6%)>L435W (75.5%)>L437W (58.7%)>wildtype (43.0%) at 0 mV (FIG. 10); these mutants happened to be fiveresidues with impaired fast inactivation. Furthermore,L435W/L437C/A438W, L437C/A438W, and A438W/I1589W mutants with multiplesubstitutions have minimal fast inactivation, and they showsignificantly enhanced slow inactivation (96.8%, 93.2%, and 88.3%,respectively).

[0077] It is surprising to find that the mutants with minimal fastinactivation express as well as the wild type in mammalian cells.Previous reports in the literature indicated that, unlike wild-type Na⁺channels, various fast-inactivation deficient mutants at the IFM motifexpressed poorly in HEK293 expression system under the same conditions.The inactivation-deficient S6 mutants of the invention are useful toolsfor future studies, including the establishment of permanent cell lines,the screening for potent open-channel blockers that block persistentopening (e.g. anti-arrhythmic agents), the ion permeation in thepersistent open channel, and the detailed studies on direct interactionsbetween drugs and the open channel.

[0078] Studies with rNav1.4-L435W/L437C/A438W demonstrated thatflecainide binds rapidly and preferentially with the open state butminimally with the resting state. Flecainide is very effective inblocking persistent late Na⁺ currents as evident from its strongtime-dependent block of maintained currents during prolongeddepolarization. Flecainide binding with the inactivated state isconsiderably slower than that with the open state by orders ofmagnitude. Once the channel is blocked by flecainide, the inactivationgate may stabilize receptor-flecainide complex, as the dissociation rateof flecainide is extremely slow and requires >1,000 s (˜17 minutes) forthe full recovery.

[0079] We first measured the Na⁺ current family at voltages ranging from−60 mV to +50 mV. We then applied 30 mM flecainide externally andmeasured the Na+ current at +50 mV for 5 ms at a 30-s interval. About50% of the peak currents were inhibited after flecainide block reachedits steady state, usually within 5-7 minutes. We then re-measured theNa⁺ current family in the presence of 30 mM flecainide and found thatthe current kinetics remained unchanged and the conductance/voltagecurves remained comparable with or without flecainide.

[0080] The steady-state inactivation of Na+ channels was measured by astandard two-pulse protocol at a test pulse of +30 mV with variousconditioning pulses ranging from −160 mV to −15 mV for 100 ms, with andwithout flecainide. There was a leftward shift of a few mV and the slopefactor appeared less steep.

[0081] We applied a voltage scanning protocol ranging from −180 mV to 0mV to determine whether distinct binding affinities of flecainide existin rNav1.4 Na+ channels. This pulse protocol consisted of a conditioningpulse at various voltages with a 10-s duration intended for drugbinding. It was originally designed to test if inactivated channels havehigher “saturable” affinities than resting channels for localanesthetics. FIG. 11 shows that flecainide at 30 μM blocks restingchannels at a constant level from −180 mV to −100 mV. The blockincreases continuously from −80 mV to −20 mV.

[0082] We measured the dose-response curve of flecainide with a 10-sconditioning pulse at −140 mV, −70 mV, and −20 mV, again at a 30-sinterval for the flecainide to reach steady state. Binding at thesethree voltages for local anesthetics generally represents the resting,closed/inactivated, and open/inactivated affinities, respectively. Themeasurements at −20 mV, −70 mV, and −140 mV provided IC₅₀ values of13.4±0.3, 21.2±0.4, and 31.9±3.0 mM, respectively. The differencebetween the high and low flecainide affinities is less than 3-fold. Inthe case of cocaine, the difference between the resting and inactivatedblock is 28-fold (250 mM vs. 9 mM).

[0083] One possibility for the rightward shift of the voltage dependenceof flecainide block is that the inactivated channels interact with thedrug rather slowly. This appeared to be the case for the development ofthe inactivated block at −50 mV with a time constant of 10.9±1.3 s. Oncedeveloped, the recovery from this inactivated block by flecainide at 100mM was also very slow with a time constant of >100 s, as if flecainidewas trapped within the channel. Unexpectedly, the amplitude of the Na+currents continued to increase during this recovery period and reached alevel that is 78% to the control amplitude without flecainide. A sameslow time course also occurred after the block was developed at +30 mV.Thus, both closed/inactivated and open/inactivated block by flecainiderecovered nearly to the level about 80% of the control value with thesame slow time course. These results indicated that the resting block at−140 mV by flecainide is much less than the block normally measured atthe 30-s interval. The estimated IC₅₀ for the resting block offlecainide in wild-type channels is 355 mM at −140 mV.

[0084] To test whether flecainide interacts with the open state of Na+channels we therefore applied repetitive pulses for channel activation.Repetitive pulses at 5 Hz for 60 pulses elicited additionaluse-dependent block of flecainide by ˜50%. The total duration ofdepolarization was 1.44 s (0.024×60). Therefore, it appears thatflecainide binds to the open state of Na+ channels with a faster ratethan that of the inactivated state since the similar long pulse did notelicit a comparable use-dependent block. This being true, keeping thechannel open persistently during depolarization enhances the block offlecainide.

[0085] To determine interactions between flecainide and the open statedirectly, we used inactivation-deficient rNav1.4-L435W/L437C/A438Wmutant channels of the invention. This mutant channel inactivatedminimally during depolarization; instead, a substantial fraction of peakcurrent was maintained. FIG. 12 shows the current families before andafter flecainide at various concentrations ranging from 0.1 to 30 μM.At+50 mV, there was a strong time-dependent block of the maintained Na+currents. This result therefore provides the direct evidence thatflecainide binds preferentially with the open state of the Na+ channeland indicates that Nav mutants of the invention can be used as tools fordetailed studies on sodium channel block.

[0086] We generated non-inactivating Na+ currents using a test pulse of+30 mV and then measured the time-dependent block of flecainide atvarious concentrations. The decay phase of the Na+ current could be wellfitted with a single exponential function and the time constant (τ) wasinverted and plotted against the corresponding concentration (FIG. 13).The on-rate and off-rate constant of flecainide with the open channelare estimated to be 11.8 μM⁻¹s⁻¹ (the slope factor) and 14.8 s⁻¹(y-intercept), respectively. The calculated dissociation constant yields0.80 mM. The IC₅₀ values for the open (estimated block at the end of thepulse) and the resting block (estimated block at the peak current) were0.61±0.07 μM and 208.3±16.9 μM, respectively. In contrast, with aconditioning pulse at −50 mV for 10 s, the IC₅₀ was 4.1±0.1 μM(estimated block at the peak current) or about 7-fold less potent thanthat of the open channel block. This suggests channel opening isrequired for the high-affinity block of flecainide. With limited channelopening around activation threshold of −50 mV, the flecainide affinityis not as high as that of the open channel block.

[0087] Repetitive pulses at 5 Hz demonstrate that flecainide produces anadditional use-dependent block in the peak current amplitude. Itappeared that this rapid phase of the use-dependent block was causeddirectly by the time-dependent block of the non-inactivating currentduring the pulse. This time-dependent block recovers little during the200-ms interpulse at −140 mV. There was also a slow inhibition of peakcurrents during repetitive pulses in inactivation-deficient channelseven without flecainide.

[0088] From the foregoing results one may conclude that: (1) Flecainideblock of the wild-type Na+ channel developed after channel activationhas a very slow recovery time course, up to 10,000 s (or ˜17 minutes) atthe holding potential of −140 mV. Any pulse protocol that activates Na+channels at a frequency as low as one per 30 s will significantlyperturb the degree of flecainide block. (2) The resting and open channelaffinities differ by ˜500-fold in the inactivation-deficient mutantchannels of the invention (0.61 μM vs. 307 μM, respectively). (3) Therecovery from the open channel block by flecainide is relatively fast at−140 mV, with a time constant of 11.2 s in inactivation-deficient mutantchannels, or several orders faster than that with intact fastinactivation in wild-type Na+ channels.

[0089] Flecainide appears to interact with the resting state of Na+channels rather weakly. At 100 mM flecainide blocks only about ˜20% ofpeak Na+ currents if the cell is not stimulated repetitively in 1,000 s.The calculated IC₅₀ for flecainide block is therefore about 400 μM. Itwill be difficult to measure this value directly in a single cell havinga wild-type Nav protein at various concentrations, since only one testpulse per 17 minutes can be applied for such dose-response assay. Incontrast, the IC₅₀ for flecainide block of inactivation-deficient mutantNa⁺ channels of the invention can be estimated directly from the peakcurrent amplitude, which yields 307±19 μM for the resting block.

[0090] Flecainide appears to be a rather pure open channel blocker withminimal interactions with resting state. Flecainide has been shown to bebeneficial for the treatment of a number of genetic diseases withmutations on the Na+ channel (e.g., Brugada et al., 1999; Windle et al.,2001). Many of these defective channels exhibit persistent late Na+currents lasting hundreds of milliseconds during prolongeddepolarization, such as in the cases of DKPQ (Bennett et al., 1995) orhyperkalemic periodic paralysis (Cannon et al., 1991). Recently,Nagatomo et al (2000) found that flecainide preferentially blocks thelate Na+ currents in the DKPQ mutant. The results above demonstrate thatflecainide indeed blocks the maintained persistent Na+ currentseffectively and rapidly. The therapeutic plasma concentration offlecainide is 0.4 to 2 μM as an antiarrhythmic agent. At thisconcentration range, a substantial fraction of the persistent latecurrent should be blocked by flecainide, which exhibits an IC₅₀ of 0.61μM for the open channel. The persistent late currents are likely morevulnerable to flecainide block as the peak currents are rapidlyinactivated and may not be blocked in time. The open-channel selectiveblockers, such as flecainide and pilsicainide have broader applicationsfor various pathological conditions that manifest an increase inpersistent late Na+ currents in the heart (Saint et al., 1992), in brain(Crill, 1996) or in muscle (Cannon, 1996) and the search for improvedagents to treat these pathological conditions is greatly advanced by thescreen of the present invention.

1 78 1 28 PRT Homo sapiens 1 Tyr Met Ile Phe Phe Val Leu Val Ile Phe LeuGly Ser Phe Tyr Leu 1 5 10 15 Ile Asn Leu Ile Leu Ala Val Val Ala MetAla Tyr 20 25 2 28 PRT Homo sapiens 2 Tyr Met Ile Phe Phe Val Leu ValIle Phe Leu Gly Ser Phe Tyr Leu 1 5 10 15 Ile Asn Leu Ile Leu Ala ValVal Ala Met Ala Tyr 20 25 3 28 PRT Homo sapiens 3 Tyr Met Ile Phe PheVal Leu Val Ile Phe Leu Gly Ser Phe Tyr Leu 1 5 10 15 Ile Asn Leu IleLeu Ala Val Val Ala Met Ala Tyr 20 25 4 28 PRT Homo sapiens 4 Tyr MetIle Phe Phe Val Val Ile Ile Phe Leu Gly Ser Phe Tyr Leu 1 5 10 15 IleAsn Leu Ile Leu Ala Val Val Ala Met Ala Tyr 20 25 5 28 PRT Homo sapiens5 Tyr Met Ile Phe Phe Met Leu Val Ile Phe Leu Gly Ser Phe Tyr Leu 1 5 1015 Val Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr 20 25 6 28 PRT Homosapiens 6 Tyr Met Ile Phe Phe Val Val Val Ile Phe Leu Gly Ser Phe TyrLeu 1 5 10 15 Val Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr 20 25 7 28PRT Homo sapiens 7 Tyr Met Ile Phe Phe Val Val Val Ile Phe Leu Gly SerPhe Tyr Leu 1 5 10 15 Ile Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr 2025 8 28 PRT Rattus sp. 8 Tyr Met Ile Phe Phe Val Val Ile Ile Phe Leu GlySer Phe Tyr Leu 1 5 10 15 Ile Asn Leu Ile Leu Ala Val Val Ala Met AlaTyr 20 25 9 28 PRT Rattus sp. 9 Tyr Met Ile Phe Phe Met Leu Val Ile PheLeu Gly Ser Phe Tyr Leu 1 5 10 15 Val Asn Leu Ile Leu Ala Val Val AlaMet Ala Tyr 20 25 10 28 PRT Rattus sp. 10 Tyr Met Ile Phe Phe Met LeuVal Ile Phe Val Gly Ser Phe Tyr Pro 1 5 10 15 Val Asn Leu Ile Leu AlaVal Val Ala Met Ala Tyr 20 25 11 28 PRT Rattus sp. 11 Tyr Met Val PhePhe Val Val Val Ile Phe Leu Gly Ser Phe Tyr Leu 1 5 10 15 Val Asn LeuIle Leu Ala Val Val Ala Met Ala Tyr 20 25 12 28 PRT Rattus sp. 12 TyrMet Val Phe Phe Met Leu Val Ile Phe Leu Gly Ser Phe Tyr Leu 1 5 10 15Val Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr 20 25 13 28 PRT Homosapiens 13 Cys Leu Thr Val Phe Met Met Val Met Val Ile Gly Asn Leu ValVal 1 5 10 15 Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe 20 25 1428 PRT Homo sapiens 14 Cys Leu Thr Val Phe Met Met Val Met Val Ile GlyAsn Leu Val Val 1 5 10 15 Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser SerPhe 20 25 15 28 PRT Homo sapiens 15 Cys Leu Ile Val Phe Met Leu Val MetVal Ile Gly Asn Leu Val Val 1 5 10 15 Leu Asn Leu Phe Leu Ala Leu LeuLeu Ser Ser Phe 20 25 16 28 PRT Homo sapiens 16 Cys Leu Leu Val Phe LeuLeu Val Met Val Ile Gly Asn Leu Val Val 1 5 10 15 Leu Asn Leu Phe LeuAla Leu Leu Leu Ser Ser Phe 20 25 17 28 PRT Rattus sp. 17 Cys Leu ThrVal Phe Leu Met Val Met Val Ile Gly Asn Leu Val Val 1 5 10 15 Leu AsnLeu Phe Leu Ala Leu Leu Leu Ser Ser Phe 20 25 18 28 PRT Rattus sp. 18Cys Leu Leu Val Phe Leu Leu Val Met Val Ile Gly Asn Leu Val Val 1 5 1015 Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe 20 25 19 28 PRT Homosapiens 19 Met Tyr Leu Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe PheThr 1 5 10 15 Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe 20 25 2028 PRT Homo sapiens 20 Met Tyr Leu Tyr Phe Val Ile Phe Ile Ile Phe GlySer Phe Phe Thr 1 5 10 15 Leu Asn Leu Phe Ile Gly Val Ile Ile Asp AsnPhe 20 25 21 28 PRT Homo sapiens 21 Met Tyr Leu Tyr Phe Val Ile Phe IleIle Phe Gly Ser Phe Phe Thr 1 5 10 15 Leu Asn Leu Phe Ile Gly Val IleIle Asp Asn Phe 20 25 22 28 PRT Homo sapiens 22 Met Tyr Leu Tyr Phe ValIle Phe Ile Ile Phe Gly Ser Phe Phe Thr 1 5 10 15 Leu Asn Leu Phe IleGly Val Ile Ile Asp Asn Phe 20 25 23 28 PRT Homo sapiens 23 Met Tyr IleTyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe Thr 1 5 10 15 Leu AsnLeu Phe Ile Gly Val Ile Ile Asp Asn Phe 20 25 24 28 PRT Homo sapiens 24Met Tyr Leu Tyr Phe Val Ile Phe Ile Ile Gly Gly Ser Phe Phe Thr 1 5 1015 Leu Asn Leu Phe Val Gly Val Ile Ile Asp Asn Phe 20 25 25 28 PRTRattus sp. 25 Met Tyr Leu Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser PhePhe Thr 1 5 10 15 Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe 20 2526 28 PRT Rattus sp. 26 Met Tyr Ile Tyr Phe Val Val Phe Ile Ile Phe GlySer Phe Phe Thr 1 5 10 15 Leu Asn Leu Phe Ile Gly Val Ile Ile Asp AsnPhe 20 25 27 28 PRT Rattus sp. 27 Met Tyr Leu Tyr Phe Val Val Phe IleIle Phe Gly Ser Phe Phe Thr 1 5 10 15 Leu Asn Leu Phe Ile Gly Val IleIle Asp Asn Phe 20 25 28 28 PRT Rattus sp. 28 Met Tyr Ile Tyr Phe ValVal Phe Ile Ile Phe Gly Gly Phe Phe Thr 1 5 10 15 Leu Asn Leu Phe ValGly Val Ile Ile Asp Asn Phe 20 25 29 28 PRT Homo sapiens 29 Gly Ile PhePhe Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Val Val 1 5 10 15 Val AsnMet Tyr Ile Ala Val Ile Leu Glu Asn Phe 20 25 30 28 PRT Homo sapiens 30Gly Ile Phe Phe Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Val Val 1 5 1015 Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn Phe 20 25 31 28 PRT Homosapiens 31 Gly Ile Phe Phe Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu ValVal 1 5 10 15 Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn Phe 20 25 3228 PRT Homo sapiens 32 Gly Ile Cys Phe Phe Cys Ser Tyr Ile Ile Ile SerPhe Leu Ile Val 1 5 10 15 Val Asn Met Tyr Ile Ala Ile Ile Leu Glu AsnPhe 20 25 33 28 PRT Homo sapiens 33 Gly Ile Leu Phe Phe Thr Thr Tyr IleIle Ile Ser Phe Leu Ile Val 1 5 10 15 Val Asn Met Tyr Ile Ala Ile IleLeu Glu Asn Phe 20 25 34 28 PRT Rattus sp. 34 Gly Ile Cys Phe Phe CysSer Tyr Ile Ile Ile Ser Phe Leu Ile Val 1 5 10 15 Val Asn Met Tyr IleAla Ile Ile Leu Glu Asn Phe 20 25 35 28 PRT Rattus sp. 35 Gly Ile LeuPhe Phe Thr Thr Tyr Ile Ile Ile Ser Phe Leu Ile Val 1 5 10 15 Val AsnMet Tyr Ile Ala Ile Ile Leu Glu Asn Phe 20 25 36 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 36 Trp IleLeu Ala Val Val Ala Met Ala Tyr 1 5 10 37 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 37 Tyr Ile Leu AlaVal Val Ala Met Ala Tyr 1 5 10 38 10 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 38 Phe Ile Leu Ala Val Val AlaMet Ala Tyr 1 5 10 39 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 39 Leu Ile Leu Trp Val Val Ala MetAla Tyr 1 5 10 40 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 40 Leu Ile Leu Tyr Val Val Ala Met Ala Tyr 15 10 41 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 41 Leu Ile Leu Phe Val Val Ala Met Ala Tyr 1 5 10 4210 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 42 Leu Ile Cys Trp Val Val Ala Met Ala Tyr 1 5 10 43 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide43 Leu Ile Cys Tyr Val Val Ala Met Ala Tyr 1 5 10 44 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 44 Leu IleCys Phe Val Val Ala Met Ala Tyr 1 5 10 45 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 45 Trp Ile Cys TrpVal Val Ala Met Ala Tyr 1 5 10 46 10 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 46 Tyr Ile Cys Tyr Val Val AlaMet Ala Tyr 1 5 10 47 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 47 Phe Ile Cys Phe Val Val Ala MetAla Tyr 1 5 10 48 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 48 Trp Ile Cys Tyr Val Val Ala Met Ala Tyr 15 10 49 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 49 Trp Ile Cys Phe Val Val Ala Met Ala Tyr 1 5 10 5010 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 50 Tyr Ile Cys Trp Val Val Ala Met Ala Tyr 1 5 10 51 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide51 Phe Ile Cys Trp Val Val Ala Met Ala Tyr 1 5 10 52 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 52 Tyr IleCys Tyr Val Val Ala Met Ala Tyr 1 5 10 53 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 53 Phe Ile Cys PheVal Val Ala Met Ala Tyr 1 5 10 54 10 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 54 Tyr Ile Cys Phe Val Val AlaMet Ala Tyr 1 5 10 55 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 55 Phe Ile Cys Tyr Val Val Ala MetAla Tyr 1 5 10 56 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 56 Leu Ile Trp Ala Val Trp Ala Met Ala Tyr 15 10 57 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 57 Leu Ile Tyr Ala Val Trp Ala Met Ala Tyr 1 5 10 5810 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 58 Leu Ile Phe Ala Val Trp Ala Met Ala Tyr 1 5 10 59 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide59 Leu Ile Leu Ala Val Trp Ala Met Ala Tyr 1 5 10 60 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 60 Met TyrIle Ala Trp Ile Leu Glu Asn Phe 1 5 10 61 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 61 Met Tyr Ile AlaTyr Ile Leu Glu Asn Phe 1 5 10 62 10 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 62 Met Tyr Ile Ala Phe Ile LeuGlu Asn Phe 1 5 10 63 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 63 Met Tyr Ile Ala Ile Trp Leu GluAsn Phe 1 5 10 64 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 64 Met Tyr Ile Ala Ile Tyr Leu Glu Asn Phe 15 10 65 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 65 Met Tyr Ile Ala Ile Phe Leu Glu Asn Phe 1 5 10 6610 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 66 Met Tyr Ile Ala Cys Ile Leu Glu Asn Phe 1 5 10 67 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide67 Met Tyr Ile Ala Ile Cys Leu Glu Asn Phe 1 5 10 68 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 68 Met TyrIle Ala Trp Trp Leu Glu Asn Phe 1 5 10 69 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 69 Met Tyr Ile AlaTyr Tyr Leu Glu Asn Phe 1 5 10 70 10 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 70 Met Tyr Ile Ala Phe Phe LeuGlu Asn Phe 1 5 10 71 18 PRT Artificial Sequence Description ofArtificial Sequence Synthetic amino acid sequence 71 Tyr Met Ile Phe PheXaa Xaa Xaa Ile Phe Leu Gly Ser Phe Tyr Leu 1 5 10 15 Xaa Asn 72 28 PRTArtificial Sequence Description of Artificial Sequence Synthetic aminoacid sequence 72 Tyr Met Ile Phe Phe Met Leu Val Ile Phe Leu Gly Ser PheTyr Leu 1 5 10 15 Val Asn Trp Ile Leu Ala Val Val Ala Met Ala Tyr 20 2573 39 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer 73 ctcatcaatc tgatctgctg ggtggtggcc atggcgtac 39 74 40DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 74 cctcatcaat tggatctgct gggtggtggc catggcgtac 40 75 41 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer75 cctcatcaat ctgatctgct gggtggtggc catggcatat g 41 76 52 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 76gctctttcta cctcatcaat tggatctgct gggtggtggc catggcatat gc 52 77 37 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer77 cctggtgaac ctgatctgct gggtggtcgc aatggcc 37 78 30 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 78ccttctacct ggtgaactgg atctgctggg 30

1. A method for assessing the potential of a compound to function as ananti-arrhythmic agent comprising: (a) providing a cell that expresses arecombinant mutant Nav1 sodium channel protein; (b) measuring a firstplateau current in said cell; (c) exposing said cell to a test compound;(d) measuring a second plateau current in said cell; and (e) comparingsaid first and second currents whereby a lower second current indicatesthat said test compound is a potential anti-arrhythmic agent; saidmutant sodium channel protein having an amino acid sequence in which oneor more amino acids among the ten amino acids occurring at the carboxyend of the S6 segments of D1 D2, D3 or D4 domains of a mammalian Nav1protein differs from the amino acid in wild-type Nav1 by substitutionwith tryptophan, phenylalanine, tyrosine or cysteine.
 2. The method ofclaim 1 wherein said mammalian Nav 1 protein is selected from Nav 1.1,Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, or Nav 1.8.
 3. Themethod of claim 2 wherein said mammalian Nav 1 protein is Nav 1.4 or Nav1.5.
 4. A method for assessing the potential of a compound as ananti-arrhythmic agent comprising: (a) providing a cell that expresses arecombinant mutant Nav1 sodium channel protein; (b) measuring a firstplateau current in said cell; (c) exposing said cell to a test compound;(d) measuring a second plateau current in said cell; and (e) comparingsaid first and second currents whereby a lower second current indicatesthat said test compound is a potential anti-arrhythmic agent; saidmutant sodium channel protein having an amino acid sequence in which atleast one amino acid chosen from amino acids 19, 21 and 22 of the S6segment of D1 and amino acids 23 and 24 of the S6 segment of the D4domain of a mammalian Nav1 protein differs from the amino acid inwild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosineor cysteine.
 5. The method of claim 4 wherein said mammalian Nav 1protein is selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5,Nav 1.6, Nav 1.7, or Nav 1.8.
 6. The method of claim 5 wherein saidmammalian Nav 1 protein is Nav 1.4 or Nav 1.5.
 7. A method for assessingthe potential of a compound as an anti-arrhythmic agent comprising: (a)providing a human cell that expresses a recombinant mutant Nav1.4 orNav1.5 sodium channel protein; (b) measuring a first plateau current insaid cell; (c) exposing said cell to a test compound; (d) measuring asecond plateau current in said cell; and (e) comparing said first andsecond currents whereby a lower second current indicates that said testcompound is a potential anti-arrhythmic agent; said mutant sodiumchannel protein having an amino acid sequence in which at least oneamino acid chosen from amino acids L435, L437, A438, I1589 and I1590 ofwild-type rNav 1.4 is replaced by tryptophan, phenylalanine or tyrosine,or in the case of L437 additionally with cysteine.
 8. A method accordingto claim 1 wherein said cell is chosen from a human embryonic kidneycell and a Chinese hamster ovary cell.
 9. A method according to claim 1wherein one or more wild-type amino acids are replaced with tryptophan.10. A method according to claim 3 wherein the mammalian Nav 1.4 or Nav1.5 is rat or human Nav 1.4 or Nav 1.5 and a leucine corresponding toL437 of rNav1.4 is replaced with cysteine.
 11. A method according toclaim 10 wherein L437 is replaced with cysteine and one or both of aleucine and an alanine corresponding to L435 and A438 respectively ofrNav1.4 are replaced with tryptophan.
 12. The method according to claim3 wherein the mammalian Nav1.4 or Nav1.5 is rat or human Nav1.4 orNav1.5.
 13. The method according to claim 12 wherein an alaninecorresponding to A438 and an isoleucine corresponding to I1589 inrNav1.4 are replaced.
 14. The method according to claim 13 wherein saidalanine and isoleucine are replaced by tryptophan.
 15. A cell comprisinga nucleic acid that encodes a recombinant mutant mammalian Nav1 protein,said mutant protein having a sequence in which one or more amino acidsamong the ten amino acids occurring at the carboxy end of the S6segments of D1, D2, D3 or D4 domains of mammalian Nav1 differs from theamino acid in wild-type Nav1 by substitution with tryptophan,phenylalanine, tyrosine or cysteine.
 16. The cell of claim 15 whereinsaid mammalian Nav 1 protein is selected from Nav 1.1, Nav 1.2, Nav 1.3,Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, Nav 1.8, or Nav 1.9.
 17. The cell ofclaim 16 wherein said mammalian Nav 1 protein is Nav 1.4 or Nav 1.5. 18.A cell comprising a nucleic acid that encodes a recombinant mutantmammalian Nav1 protein, said mutant protein having an amino acidsequence in which at least one amino acid chosen from amino acids 19, 21and 22 of the S6 segment of D1 and amino acids 23 and 24 of the S6segment of the D4 domain of mammalian Nav1 differs from the amino acidin wild-type Navl by substitution with tryptophan, phenylalanine,tyrosine or cysteine.
 19. The cell of claim 18 wherein said mammalianNav 1 protein is selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav1.5, Nav 1.6, Nav 1.7, Nav 1.8, or Nav 1.9.
 20. The cell of claim 19wherein said mammalian Nav 1 protein is Nav 1.4 or Nav 1.5.
 21. A humancell comprising a nucleic acid that encodes a mutant mammalian Nav 1.4or Nav 1.5 protein, said mutant sodium channel protein having an aminoacid sequence in which at least one amino acid chosen from amino acidsL435, L437, A438, I1589 and I1590 of wild-type rat Nav1.4 is replaced bytryptophan, phenylalanine or tyrosine, or in the case of L437additionally with cysteine.
 22. A cell according of claim 15 whereinsaid mutant sodium channel protein gives rise to sodium channelsexhibiting plateau currents of greater than 1 nanoamp.
 23. An isolatednucleic acid comprising a nucleotide sequence that codes for a mutantmammalian Nav1 protein, said mutant protein having an amino acidsequence in which one or more amino acids among the ten amino acidsoccurring at the carboxy end of the S6 segments of D1, D2, D3 or D4domains of mammalian Nav1 differs from the amino acid in wild-type Nav1by substitution with tryptophan, phenylalanine, tyrosine or cysteine.24. The isolated nucleic acid of claim 23 wherein said mammalian Nav 1protein is selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5,Nav 1.6, Nav 1.7, Nav 1.8 or Nav 1.9.
 25. The isolated nucleic acid ofclaim 24 wherein said mammalian Nav 1 protein is Nav 1.4 or Nav 1.5. 26.An isolated nucleic acid comprising a nucleotide sequence that codes fora mutant mammalian Nav1.4 or Nav1.5 protein, said mutant protein havingan amino acid sequence in which at least one amino acid chosen fromamino acids 19, 21 and 22 of the S6 segment of D1 and amino acids 23 and24 of the S6 segment of the D4 domain of said mutant mammalian Nav 1.4or Nav 1.5 differs from the amino acid in wild-type mammalian Nav 1.4 orNav 1.5 by substitution with tryptophan, phenylalanine, tyrosine orcysteine.
 27. An isolated nucleic acid comprising a nucleotide sequencethat codes for a rat or human Nav1.4 or Nav1.5 protein in which one ormore amino acids corresponding to wild-type amino acids L435, L437,A438, I1589 and I1590 of rNav 1.4 is replaced with tryptophan,phenylalanine or tyrosine, or in the case of L437 additionally withcysteine.
 28. A isolated functional sodium channel protein comprising afirst amino acid sequence chosen from: WILAVVAMAY SEQ ID NO.: 36YILAVVAMAY SEQ ID NO.: 37 FILAVVAMAY SEQ ID NO.: 38 LILWVVAMAY SEQ IDNO.: 39 LILYVVAMAY SEQ ID NO.: 40 LILFVVAMAY SEQ ID NO.: 41 LICWVVAMAYSEQ ID NO.: 42 LICYVVAMAY SEQ ID NO.: 43 LICFVVAMAY SEQ ID NO.: 44WICWVVAMAY SEQ ID NO.: 45 YICYVVAMAY SEQ ID NO.: 46 FICFVVAMAY SEQ IDNO.: 47 WICYVVAMAY SEQ ID NO.: 48 WICFVVAMAY SEQ ID NO.: 49 YICWVVAMAYSEQ ID NO.: 50 FICWVVAMAY SEQ ID NO.: 51 YICYVVAMAY SEQ ID NO.: 52FICFVVAMAY SEQ ID NO.: 53 YICFVVAMAY SEQ ID NO.: 54 FICYVVAMAY SEQ IDNO.: 55 LIWAVWAMAY SEQ ID NO.: 56 LIYAVWAMAY SEQ ID NO.: 57 LIFAVWAMAYSEQ ID NO.: 58 LILAVWAMAY SEQ ID NO.: 59 MYIAWILENF SEQ ID NO.: 60MYIAYILENF SEQ ID NO.: 61 MYIAFILENF SEQ ID NO.: 62 MYIAIWLENF SEQ IDNO.: 63 MYIAIYLENF SEQ ID NO.: 64 MYIAIFLENF SEQ ID NO.: 65 MYIACILENFSEQ ID NO.: 66 MYIAICLENF SEQ ID NO.: 67 MYIAWWLENF SEQ ID NO.: 68MYIAYYLENF SEQ ID NO.: 69 MYIAFFLENF SEQ ID NO.: 70


29. A functional sodium channel protein comprising first and secondamino acid sequences, said first amino acid sequence chosen from:WILAVVAMAY SEQ ID NO.: 36 LILWVVAMAY SEQ ID NO.: 39 LICWVVAMAY SEQ IDNO.: 42 WICWVVAMAY, SEQ ID NO.: 45 and LILAVWAMAY SEQ ID NO.: 59

and said second amino acid sequence chosen from: MYIAWILENF SEQ ID NO.:60 MYIAIWLENF SEQ ID NO.: 63 MYIACILENF SEQ ID NO.: 66 MYIAICLENF, SEQID NO.: 67 and MYIAWWLENF. SEQ ID NO.: 68


30. A functional recombinant sodium channel protein according to claim18 additionally comprising a second amino acid sequenceYMIFFX^(a)X^(b)X^(c)IFLGSFYLX^(d)N (SEQ ID NO. 71) immediately adjacentand amino-terminal to said first amino acid sequence, wherein X^(a) ischosen from V and M; X^(b) is chosen from L and V; X^(c) is chosen from1 and V; and X^(d) is chosen from I and V.
 31. A screen for assessingthe potential of a compound to treat a pathological condition manifestedby an increased late sodium current in a heart comprising: (a) providinga cell that expresses a recombinant mutant Nav1 sodium channel protein;(b) measuring a first plateau current in said cell; (c) exposing saidcell to a test compound; (d) measuring a second plateau current in saidcell; and (e) comparing said first and second currents whereby a lowersecond current indicates that said test compound is a potentialanti-arrhythmic agent; said mutant sodium channel protein having anamino acid sequence in which one or more amino acids among the ten aminoacids occurring at the carboxy end of the S6 segments of D1, D2, D3 orD4 domains of mammalian Nav1 differs from the amino acid in wild-typeNav1 by substitution with tryptophan, phenylalanine, tyrosine orcysteine.
 32. The method of claim 31 wherein said mammalian Nav1 proteinis selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6,Nav 1.7, or Nav 1.8.
 33. The method of claim 32 wherein said mammalianNav 1 protein is Nav 1.4 or Nav 1.5.
 34. A screen for assessing thepotential of a compound to treat a pathological condition manifested byan increased late sodium current in a heart comprising: (a) providing acell that expresses a mutant Nav1 sodium channel protein; (b) measuringa first plateau current in said cell; (c) exposing said cell to a testcompound; (d) measuring a second plateau current in said cell; and (e)comparing said first and second currents whereby a lower second currentindicates that said test compound is a potential anti-arrhythmic agent;said mutant sodium channel protein having an amino acid sequence inwhich at least one amino acid chosen from amino acids 19, 21 and 22 ofthe S6 segment of D1 and amino acids 23 and 24 of the S6 segment of theD4 domain of mammalian Nav1.4 or Nav1.5 differs from the amino acid inwild-type NavI by substitution with tryptophan, phenylalanine, tyrosineor cysteine.
 35. The method of claim 34 wherein said mammalian Nav1protein is selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5,Nav 1.6, Nav 1.7, or Nav 1.8.
 36. The method of claim 35 wherein saidmammalian Nav 1 protein is Nav 1.4 or Nav 1.5.
 37. A screen forassessing the potential of a compound to treat a pathological conditionmanifested by an increased late sodium current in a heart comprising:(a) culturing a human cell that produces mutant Nav 1.4 or Nav 1.5sodium channel protein; (b) measuring a first plateau current in saidcell; (c) exposing said cell to a test compound; (d) measuring a secondplateau current in said cell; and (e) comparing said first and secondcurrents whereby a lower second current indicates that said testcompound is a potential anti-arrhythmic agent; said mutant sodiumchannel protein having an amino acid sequence in which at least oneamino acid chosen from amino acids L435, L437, A438, I1589 and I1590 ofwild-type rNav 1.4 is replaced by tryptophan, phenylalanine or tyrosine,or in the case of L437 additionally with cysteine.
 38. A screenaccording to claim 34 wherein said cell is chosen from a human embryonickidney cell and a Chinese hamster ovary cell.
 39. A screen according toclaim 34 wherein one or more wild-type amino acids are replaced withtryptophan.
 40. A screen according to claim 34 wherein the mammalianNav1.4 or Nav1.5 is rat or human Nav1.4 or Nav1.5 and a leucinecorresponding to L437 of rNav 1.4 is replaced with cysteine.
 41. Ascreen according to claim 40 wherein L437 is replaced with cysteine andone or both of a leucine and an alanine corresponding to L435 and A438respectively of rNav1.4 are replaced with tryptophan.
 42. A screenaccording to claim 34 wherein the mammalian Nav1.4 or Nav1.5 is rat orhuman Nav1.4 or Nav1.5.
 43. A screen according to claim 42 wherein analanine corresponding to A438 and an isoleucine corresponding to I1589in rNav1.4 are replaced.
 44. A screen according to claim 43 wherein saidalanine and isoleucine are replaced by tryptophan.
 45. A screenaccording to claim 34 wherein said mutant sodium channel protein givesrise to sodium channels exhibiting plateau currents of greater than 1nanoamp.